Abstract
Purpose :
In dry eye syndrome, hyperosmolarity is one of the main factors inducing ocular surface inflammation. The objective of this work is to decipher the cellular and molecular cascades in cornea and conjunctiva, which are involved in triggering inflammation, by focusing particularly on the nuclear factor of activated T-cells 5 (NFAT5) and its implication.
Methods :
HCE (Human Corneal Epithelial) and WKD (Wong-Kilbourne derivative of Chang conjunctival cell) were cultured in normal medium or hyperosmolar medium (addition of 70 mM or 90 mM NaCl). Quantitative reverse transcription (RT-qPCR) and Western-Blotting assays were performed to analyze NFAT5 expression in corneal and conjunctival cells regarding medium (normal or hyperosmolar) and exposure times (6h, 12h and 24h). The activation of NFAT5 signaling pathway by hyperosmolarity cells was tested using Luciferase NFAT5 reporter assay system. Furthermore, cytokines expression analysis were performed by RT-qPCR and Multiplex assay, respectively at 24h and 48h, in both cells. Finally, the implication of NFAT5 in inflammation (cytokines production and release) was confirmed using the transfection of siRNA NFAT5.
Results :
Our result demonstrate the NFAT5 induction and activation in both HCE and WKD in hyperosmolar context. Furthermore, among the inflammatory messages induced in hyperosmolar stress conditions, MCP1 levels were significantly reduced after inhibition of NFAT5 in WKD cells, and reduction of IL1 beta, TNF alpha and MCP1 levels in HCE cells.
Conclusions :
These in vitro results highlight the major role of NFAT5 in the hyperosmolarity pro-inflammatory consequences but looks to be cell type specific between conjunctival and corneal epithelial cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.